The secretory pathway of eukaryotic cells consists of a series of distinct organelles between which proteins are moved in a vectorial manner. Despite the extensive movement of proteins and lipids into and out of them, each organelle maintains a set of proteins which define its unique structural and functional properties. Maintaining these resident proteins appears to be achieved at least in part by use of retrieval signals that target a protein back to its correct location along a retrograde transport route. We have previously identified a double lysine motif at the COOH terminus of type I and III membrane proteins that confers residency in the ER. Likewise a C-terminal tetra-peptide KDEL maintains soluble proteins in the lumen of the ER. We have recently shown that the double lysine motif, like the KDEL motif is a retrieval signal directing the transport of proteins back to the ER. Preliminary data has been obtained that a 15 amino acid sequence at the amino terminus of a type II ER membrane protein, the p33 form of invariant chain (Ii), is necessary for the targeting of this protein to the ER. The major goal of this proposal is to characterize this targeting motif by extensive site directed mutagenesis and to determine if it maintains other type II membrane proteins in the ER when transplanted. Next we will investigate whether this motif directs retrieval to the ER in a manner analogous to the double lysine motif. The types and rates of post-translational modifications received by marker proteins with appended double lysine and KDEL motifs suggest these proteins may well be retrieved from multiple locations throughout the Golgi stack in a manner predicted by the distillation hypothesis (Rothman, 1981). To investigate this we will compare and contrast the location of marker proteins tagged with KDEL, double lysine or Iip33 targeting motifs to each other and to known residents of the various organelles by use of subcellular fractionation and immunofluorescence and electron microscopy. In this way we will chart the retrieval path way directed by each of these motifs, and evaluate the role the various post-ER compartments play in these processes. Our long term goal is to achieve a molecular description of the mechanism by which membrane proteins with these ER targeting motifs are recognized and retrieved to the ER. To this end we will begin work to devise a genetic screen to identify yeast mutants that are deficient in this process. Complimentation of such mutants should allow us to isolate of a number of components involved in this sorting process, and thereby greatly improve our understanding of how the organelles of the secretory pathway are established and maintained.